Prox 1 in eye degeneration and sensory organ compensation during development and evolution of the cavefish Astyanax

We have investigated expression of the homeobox gene Prox 1 during eye degeneration and sensory organ compensation in cavefish embryos. The teleost Astyanax mexicanus consists of sighted surface-dwelling forms (surface fish) and several populations of blind cave-dwelling forms (cavefish), which have evolved independently. Eye formation is initiated during cavefish development, but the lens vesicle undergoes apoptosis, and the eye subsequently arrests and degenerates. The requirement of Prox 1 for lens fiber differentiation and γ-crystallin expression in the mouse suggests that changes in the expression of this gene could be involved in cavefish eye degeneration. Surface fish and cavefish embryos stained with a Prox 1 antibody showed Prox 1 expression in the lens, neuroretina, myotomes, heart, hindbrain, and gut, as reported in other vertebrates. We found that Prox 1 expression is not altered during cavefish lens development. Prox 1 protein was detected in the lens vesicle as soon as it formed and persisted until the time of lens degeneration in each cavefish population. The cavefish lens vesicle was also shown to express a γ-crystallin gene, suggesting that Prox 1 is functional in cavefish lens development. In addition to the tissues described above, Prox 1 is expressed in developing taste buds and neuromasts in cavefish, which are enhanced to compensate for blindness. It is concluded that the Prox 1 gene is not involved in lens degeneration, but that expansion of the Prox 1 expression domain occurs during taste bud and neuromast development in cavefish. Received: 31 July 1999 / Accepted: 8 November 1999     

Early and late changes in Pax6 expression accompany eye degeneration during cavefish development

We have compared Pax6 expression during embryonic development in the eyed surface form (surface fish) and several different eyeless cave forms (cavefish) of the teleost Astyanax mexicanus. Despite lacking functional eyes as adults, cavefish embryos form small optic primordia, which later arrest in development and show various degrees of eye degeneration. The pattern of Pax6 mRNA expression was modified early and late during cavefish development. In early surface fish embryos, two bilateral Pax6 expression domains are present in the anterior neural plate, which extend across the midline and fuse to form the forebrain and optic primordia. In cavefish embryos, these Pax6 domains are diminished in size and remain separated, resulting in an anterior gap in Pax6 expression and presumably the formation of smaller optic primordia. The anterior gap in Pax6 expression was confirmed by double staining for Pax6 and distalless-3 mRNA, which marks the anterior margin of the neural plate and is unaltered in cavefish. Similar anterior gaps in Pax6 expression occurred in independently derived cavefish populations, suggesting that they are important in eye degeneration. Later during surface fish development, Pax6 protein is expressed in the cornea, lens, and ganglion and amacrine cells of the neural retina. Pax6 expression was gradually reduced during cavefish lens development, concomitant with lens arrest and degeneration, and was absent in the corneal epithelium, which does not differentiate in cavefish. In contrast, Pax6 expression in the retinal ganglion and amarcine cells is unmodified in cavefish, despite retarded retinal development. The results suggest that changes in Pax6 expression are involved in the evolution of cavefish eye degeneration.     

Development and evolution of craniofacial patterning is mediated by eye-dependent and -independent processes in the cavefish Astyanax

We studied the development and evolution of craniofacial features in the teleost fish, Astyanax mexicanus. This species has an eyed surface dwelling form (surface fish) and many different cave dwelling forms (cavefish) with various degrees of reduced eyes and pigmentation. The craniofacial features we examined are the tooth-bearing maxillary bones, the nasal and antorbital bones, the circumorbital bones, and the opercular bones, all of which show evolutionary modifications in different cavefish populations. Manipulations of eye formation by transplantation of the embryonic lens, by lentectomy, or by removing the optic vesicle showed that eye-dependent and -independent processes change both the surface fish and cavefish craniofacial skeletons. The size of the olfactory pits, which the nasal and antorbital bones define, and the size and positioning of the circumorbital bones were found to correlate with eye development. For the six suborbital bones (SO1-6), the relationship with the developing eye appears to be due to ossification initiated from foci in the suborbital canal of cranial neuromasts, whose patterning is also highly correlated with the presence or absence of an eye. By contrast, we found that the number of maxillary teeth, the number of SO3 bone elements, the positioning of SO4-6 with respect to the opercular bone, and the shape of the opercular bone are not dependent on eye formation and vary among different cavefish populations. The results suggest that evolution of the cavefish craniofacial skeleton is controlled by multiple developmental events, some a direct consequence of eye degeneration and others unrelated to loss of the eye.     

Adaptive evolution of eye degeneration in the Mexican blind cavefish

The evolutionary mechanisms responsible for eye degeneration in cave-adapted animals have not been resolved. Opposing hypotheses invoking neural mutation or natural selection, each with certain genetic and developmental expectations, have been advanced to explain eye regression, although little or no experimental evidence has been presented to support or reject either theory. Here we review recent developmental and molecular studies in the teleost Astyanax mexicanus, a single species consisting of a sighted surface-dwelling form (surface fish) and many blind cave-dwelling forms (cavefish), which shed new light on this problem. The manner of eye development and degeneration, the ability to experimentally restore eyes, gene expression patterns, and comparisons between different cavefish populations all provide important clues for understanding the evolutionary forces responsible for eye degeneration. A key discovery is that Hedgehog midline signaling is expanded and inhibits eye formation by inducing lens apoptosis in cavefish embryos. Accordingly, eyes could have been lost by default as a consequence of natural selection for constructive traits, such as feeding structures, which are positively regulated by Hh signaling. We conclude from these studies that eye degeneration in cavefish may be caused by adaptive evolution and pleiotropy.     

Blind cavefish and heat shock protein chaperones: a novel role for hsp90alpha in lens apoptosis

Lens apoptosis plays a central role in cavefish eye degeneration. Heat shock proteins (hsps) can regulate apoptosis; therefore, we examined the relationship between constitutive hsp70 and hsp90 expression and lens apoptosis. The model system is Astyanax mexicanus, a teleost species consisting of an eyed surface-dwelling (surface fish) form and numerous blind cave-dwelling (cavefish) forms. Optic primordia are formed in the cavefish embryo but they subsequently undergo lens apoptosis, arrest in development and degenerate. Astyanax hsp90 and hsp70 DNAs were isolated to use as probes to compare gene expression during surface fish and cavefish development. Hsp90beta, which encodes one of two hsp90 isoforms, was not expressed in the surface fish or cavefish lens, whereas hsp70 was expressed in the lens of both forms, suggesting that neither is directly involved in lens apoptosis. In contrast, hsp90alpha, the other hsp90 isoform, was expressed in the cavefish but not the surface fish lens. Hsp90alpha expression peaked shortly before the beginning of lens apoptosis in three convergent cavefish populations, suggesting a close relationship with lens apoptosis. The absence of hsp90beta in the lens allowed us to use geldanamycin and radicicol, specific inhibitors of hsp90 chaperone function, to determine whether lens cell death requires hsp90alpha expression. Both inhibitors blocked TUNEL labeling in the cavefish lens, suggesting that hsp90alpha is required for apoptosis. In contrast to their effects on the lens, these inhibitors induced TUNEL labeling in the surface epidermis, presumably due to effects on hsp90beta function, implying that the two-hsp90 isoforms may have contrasting roles in cell survival. We conclude that hsp90alpha plays a novel role in lens apoptosis and cavefish eye degeneration.     

The lens controls cell survival in the retina: Evidence from the blind cavefish Astyanax

The lens influences retinal growth and differentiation during vertebrate eye development but the mechanisms are not understood. The role of the lens in retinal growth and development was studied in the teleost Astyanax mexicanus, which has eyed surface-dwelling (surface fish) and blind cave-dwelling (cavefish) forms. A lens and laminated retina initially develop in cavefish embryos, but the lens dies by apoptosis. The cavefish retina is subsequently disorganized, apoptotic cells appear, the photoreceptor layer degenerates, and retinal growth is arrested. We show here by PCNA, BrdU, and TUNEL labeling that cell proliferation continues in the adult cavefish retina but the newly born cells are removed by apoptosis. Surface fish to cavefish lens transplantation, which restores retinal growth and rod cell differentiation, abolished apoptosis in the retina but not in the RPE. Surface fish lens deletion did not cause apoptosis in the surface fish retina or affect RPE differentiation. Neither lens transplantation in cavefish nor lens deletion in surface fish affected retinal cell proliferation. We conclude that the lens acts in concert with another optic component, possibly the RPE, to promote retinal cell survival. Accordingly, deficiency in both optic structures may lead to eye degeneration in cavefish.     

Early Lens Ablation Causes Dramatic Long-Term Effects on the Shape of Bones in the Craniofacial Skeleton of Astyanax mexicanus

The Mexican tetra, Astyanax mexicanus, exists as two morphs of a single species, a sighted surface morph and a blind cavefish. In addition to eye regression, cavefish have an increased number of taste buds, maxillary teeth and have an altered craniofacial skeleton compared to the sighted morph. We investigated the effect the lens has on the development of the surrounding skeleton, by ablating the lens at different time points during ontogeny. This unique long-term study sheds light on how early embryonic manipulations on the eye can affect the shape of the adult skull more than a year later, and the developmental window during which time these effects occur. The effects of lens ablation were analyzed by whole-mount bone staining, immunohistochemisty and landmark based morphometric analyzes. Our results indicate that lens ablation has the greatest impact on the skeleton when it is ablated at one day post fertilisation (dpf) compared to at four dpf. Morphometric analyzes indicate that there is a statistically significant difference in the shape of the supraorbital bone and suborbital bones four through six. These bones expand into the eye orbit exhibiting plasticity in their shape. Interestingly, the number of caudal teeth on the lower jaw is also affected by lens ablation. In contrast, the shape of the calvariae, the length of the mandible, and the number of mandibular taste buds are unaltered by lens removal. We demonstrate the plasticity of some craniofacial elements and the stability of others in the skull. Furthermore, this study highlights interactions present between sensory systems during early development and sheds light on the cavefish phenotype.     

Retinal homeobox genes and the role of cell proliferation in cavefish eye degeneration

The teleost Astyanax mexicanus exhibits eyed surface dwelling (surface fish) and blind cave dwelling (cavefish) forms. Despite lacking functional eyes as adults, cavefish embryos form eye primordia, which later arrest in development, degenerate and sink into the orbit. We are comparing the expression patterns of various eye regulatory genes during surfacefish and cavefish development to determine the cause of eye degeneration. Here we examine Rx and Chx/Vsx family homeobox genes, which have a major role in cell proliferation in the vertebrate retina. We isolated and sequenced a full-length RxcDNA clone (As-Rx1) and part of a Chx/Vsx(As-Vsx2) gene, which appear to be most closely related to the zebrafish Rx1 and Alx/Vsx2 genes respectively. In situ hybridization shows that these genes have similar but non-identical expression patterns during Astyanax eye development. Expression is first detected in the optic vesicle, then throughout the presumptive retina of the optic cup, and finally in the ciliary marginal zone (CMZ), the region of the growing retina where most new retinoblasts are formed. In addition, As-Rx1 is expressed in the outer nuclear layer (ONL) of the retina, which contains the photoreceptor cells, and As-Vsx2 is expressed in the inner nuclear layer, probably in the bipolar cells. With the exception of reduced As-Rx-1 expression in the ONL, the As-Rx1 and As-Vsx2 expression patterns were unchanged in the developing retina of two different cavefish populations, suggesting that cell proliferation is not inhibited. These results were confirmed by using PCNA and BrdU markers for retinal cell division. We conclude that the CMZ is active in cell proliferation long after eye growth is diminished and is therefore not the major cause of eye degeneration.     

Conservation of retinal circadian rhythms during cavefish eye degeneration

Regressive evolution of morphological features is a common evolutionary event. However, the relationship between structural degeneration and loss of physiological function is often unclear because the ancestral and derived states of a character are usually not available for comparison. Here, we report studies on retinomotor rhythms during development of the blind cavefish Astyanax mexicanus, a single teleost species consisting of a sighted surface-dwelling form (surface fish) and several blind cave-dwelling (cavefish) forms. The eyed and blind forms of Astyanax diverged from a common sighted ancestor within the past million years. Despite the absence of functional eyes in cavefish adults, optic primordia are formed in embryos, but then gradually arrest in development, degenerate, and sink into the orbits. Although a layered retina is formed in cavefish embryos, it is deficient in photoreceptor cells, and in some cases the retinal pigment epithelium has lost its pigmentation. We show that the capacity to exhibit light-entrained retinomotor rhythms has been conserved in the degenerating embryonic eyes of two different Astyanax cavefish populations. The results indicate that loss of circadian retinal function does not precede and is therefore not required for eye degeneration in the blind cavefish.     

Modularity and sense organs in the blind cavefish, Astyanax mexicanus

SUMMARY Mexican tetra (Astyanax mexicanus) exist as two morphs: a sighted (surface) form and a blind (cavefish) form. In the cavefish, some modules are lost, such as the eye and pigment modules, whereas others are expanded, such as the taste bud and cranial neuromast modules. We suggest that modularity can be viewed as being nested in a manner similar to Baupläne so that modules express unique sets of genes, cells, and processes. In terms of evolution, we conclude that natural selection can act on any of these hierarchical levels within modules or on all the sensory modules as a whole. We discuss interactions within and between modules with reference to the blind cavefish from both genetic and developmental perspectives. The cavefish represents an illuminating example of module interaction, uncoupling of modules, and module expansion.     

To see or not to see: evolution of eye degeneration in mexican blind cavefish

The evolutionary mechanisms responsible for the loss of eyesin cave animals are still unresolved. Hypotheses invoking naturalselection or neutral mutation have been advanced to explaineye regression. Here we describe comparative molecular and developmentalstudies in the teleost Astyanax mexicanus that shed new lighton this problem. A. mexicanus is a single species consistingof a sighted surface-dwelling form (surface fish) and many blindcave-dwelling forms (cavefish) from different caves. We firstreview the evolutionary relationships of Astyanax cavefish populationsand conclude that eye degeneration may have evolved multipletimes. We then compare the mechanisms of eye degeneration indifferent cavefish populations. We describe the results of experimentsshowing that programmed cell death of the lens plays a key rolein controlling eye degeneration in these cavefish populations.We also show that Pax6 gene expression and fate determinationin the optic primordia are modified similarly in different cavefishpopulations, probably due to hyperactive midline signaling.We discuss the contributions of the comparative developmentalapproach toward resolving the evolutionary mechanisms of eyedegeneration. A new hypothesis is presented in which both naturalselection and neutral mutation are proposed to have roles incavefish eye degeneration.     

Evolution of Eye Regression in the Cavefish Astyanax: Apoptosis and the Pax-6 Gene

SYNOPSIS. The eye is an extraordinary organ in terms of itsdevelopment and evolution. In cave animals, the eye is sometimesreduced or eliminated as a consequence of adaptation to lifein perpetual darkness. We have used the characid teleost Astyanaxmexicanus as a model system to investigate the mechanisms ofeye degeneration during the evolution of a cave vertebrate.Eyed surface populations of Astyanax entered caves during thePleistocene, and their descendants lost their eyes and pigmentation.Astyanax populations exhibiting various degrees of eye regressionhave been reported in 29 Mexican caves. Surface populationswith characteristics of the ancestral stock still exist in thevicinity of these caves. Thus, Astyanax represents one of thefew instances in which the ancestral (surface fish) and thederived (cavefish) developmental modes are extant and availablefor comparative studies. The cavefish embryo develops an opticprimordium consisting of a lens vesicle and optic cup but therudimentary eye arrests in development and degenerates. Herewe report that eye degeneration is accompanied by extensiveapoptosis and downregulation of the Pax-6 gene in the developinglens. The results suggest that alterations in lens developmentare important factors in eye regression during cavefish evolution.     

A large-scale in situ screen provides molecular evidence for the induction of eye anterior segment structures by the developing lens

The anterior segment of the vertebrate eye includes the cornea, iris, ciliary body, trabecular meshwork, and lens. Although malformations of these structures have been implicated in many human eye diseases, little is known about the molecular mechanisms that control their development. To identify genes involved in anterior segment formation, we developed a large-scale in situ hybridization screen and examined the spatial and temporal expression of over 1000 genes during eye development. This screen identified 62 genes with distinct expression patterns in specific eye structures, including several expressed in novel patterns in the anterior segment. Using these genes as developmental markers, we tested for the presence of inductive signals that control the differentiation of anterior segment tissues. Organ culture recombination experiments showed that a chick lens is capable of inducing the expression of markers of the presumptive iris and ciliary body in the developing mouse neural retina. The inducing activity from the lens acts only over short ranges and is present at multiple stages of eye development. These studies provide molecular evidence that an evolutionarily conserved signal from the lens controls tissue specification in the developing optic cup.     

Probing teleost eye development by lens transplantation

Experimental manipulation and other lines of evidence indicate that the lens plays a prominent role in the growth and differentiation of the vertebrate eye. Here we describe a lens transplantation method for studying the role of the lens in teleost eye development. The method involves three steps: (1) preparing embryos for the operations by embedding them in agar, (2) microsurgery with tungsten needles to remove the lens from a donor embryo and insert it into the optic cup of a host embryo lacking its own lens, and (3) a recovery period allowing surface ectoderm to close over the wound left by insertion of the lens into the host embryo. A movie illustrating the method can be found at http://www.life.umd.edu/labs/jeffery. A troubleshooting guide and summary of assays for evaluating the development of the transplanted lens and its effects on other eye parts, including the retina, are presented. Finally, some current applications of the lens transplantation method are briefly described: (1) determination of the autonomy of zebrafish lens mutants and (2) investigation of the role of the lens in eye degeneration in the cavefish Astyanax. The transplantation method will help characterize the mechanisms through which vertebrate eye development is regulated by the lens.     

Developmental analysis of ocular morphogenesis in alpha A-crystallin/diphtheria toxin transgenic mice undergoing ablation of the lens.

The role of the lens in early eye development was examined in transgenic mice carrying the cytotoxic diphtheria toxin A gene driven by hamster alpha A-crystallin promoter sequences. Mice hemizygous for this construct are microphthalmic and contain a vacuolated and highly disorganized lens, whereas adult homozygous mice are completely ablated of the lens and lack a pupil, aqueous and posterior chamber, vitreous humor, iris, and ciliary body and show extensive convolution of the sensory retina. Developmental analysis of animals homozygous for the transgene revealed that the optic cup and lens vesicle form normally and that ablation of the lens occurs as a gradual degenerative process beginning between Days 12 and 13 of gestation. Degeneration of the lens vesicle coincides with retarded growth and development of the neuroretina, sclera, and cornea. The anterior lip of the optic cup fails to differentiate into the normal epithelium of the iris and ciliary body and the vitreous body does not develop. Although the retinal layers apparently form normally, retinal folding becomes prominent following lens degeneration. These results suggest that development of a functional lens from Embryonic Day 12.5 onward is critical for formation of the ciliary epithelium, iris, and vitreous body, as well as for appropriate growth, development, and maintenance of morphology of the retina, cornea, sclera, and optic nerve. Our results also provide information on the time course of DT-A-mediated cell destruction in vivo and are discussed in context with previous lens ablation studies and the importance of developmental analysis for interpretation of the extent to which morphogenetic aberrations are concurrent with or secondary to genetic ablation of the target tissue.     

Restoring eye size in Astyanax mexicanus blind cavefish embryos through modulation of the Shh and Fgf8 forebrain organising centres

The cavefish morph of the Mexican tetra (Astyanax mexicanus) is blind at adult stage, although an eye that includes a retina and a lens develops during embryogenesis. There are, however, two major defects in cavefish eye development. One is lens apoptosis, a phenomenon that is indirectly linked to the expansion of ventral midline sonic hedgehog (Shh) expression during gastrulation and that induces eye degeneration. The other is the lack of the ventral quadrant of the retina. Here, we show that such ventralisation is not extended to the entire forebrain because fibroblast growth factor 8 (Fgf8), which is expressed in the forebrain rostral signalling centre, is activated 2 hours earlier in cavefish embryos than in their surface fish counterparts, in response to stronger Shh signalling in cavefish. We also show that neural plate patterning and morphogenesis are modified in cavefish, as assessed by Lhx2 and Lhx9 expression. Inhibition of Fgf receptor signalling in cavefish with SU5402 during gastrulation/early neurulation mimics the typical surface fish phenotype for both Shh and Lhx2/9 gene expression. Fate-mapping experiments show that posterior medial cells of the anterior neural plate, which lack Lhx2 expression in cavefish, contribute to the ventral quadrant of the retina in surface fish, whereas they contribute to the hypothalamus in cavefish. Furthermore, when Lhx2 expression is rescued in cavefish after SU5402 treatment, the ventral quadrant of the retina is also rescued. We propose that increased Shh signalling in cavefish causes earlier Fgf8 expression, a crucial heterochrony that is responsible for Lhx2 expression and retina morphogenesis defect.     

Evolution of an adaptive behavior and its sensory receptors promotes eye regression in blind cavefish

Background

How and why animals lose eyesight during adaptation to the dark and food-limited cave environment has puzzled biologists since the time of Darwin. More recently, several different adaptive hypotheses have been proposed to explain eye degeneration based on studies in the teleost Astyanax mexicanus, which consists of blind cave-dwelling (cavefish) and sighted surface-dwelling (surface fish) forms. One of these hypotheses is that eye regression is the result of indirect selection for constructive characters that are negatively linked to eye development through the pleiotropic effects of Sonic Hedgehog (SHH) signaling. However, subsequent genetic analyses suggested that other mechanisms also contribute to eye regression in Astyanax cavefish. Here, we introduce a new approach to this problem by investigating the phenotypic and genetic relationships between a suite of non-visual constructive traits and eye regression.

Results

Using quantitative genetic analysis of crosses between surface fish, the Pachón cavefish population and their hybrid progeny, we show that the adaptive vibration attraction behavior (VAB) and its sensory receptors, superficial neuromasts (SN) specifically found within the cavefish eye orbit (EO), are genetically correlated with reduced eye size. The quantitative trait loci (QTL) for these three traits form two clusters of congruent or overlapping QTL on Astyanax linkage groups (LG) 2 and 17, but not at the shh locus on LG 13. Ablation of EO SN in cavefish demonstrated a major role for these sensory receptors in VAB expression. Furthermore, experimental induction of eye regression in surface fish via shh overexpression showed that the absence of eyes was insufficient to promote the appearance of VAB or EO SN.

Conclusions

We conclude that natural selection for the enhancement of VAB and EO SN indirectly promotes eye regression in the Pachón cavefish population through an antagonistic relationship involving genetic linkage or pleiotropy among the genetic factors underlying these traits. This study demonstrates a trade-off between the evolution of a non-visual sensory system and eye regression during the adaptive evolution of Astyanax to the cave environment.

     

Evidence for multiple genetic forms with similar eyeless phenotypes in the blind cavefish, Astyanax mexicanus

A diverse group of animals has adapted to caves and lost their eyes and pigmentation, but little is known about how these animals and their striking phenotypes have evolved. The teleost Astyanax mexicanus consists of an eyed epigean form (surface fish) and at least 29 different populations of eyeless hypogean forms (cavefish). Current alternative hypotheses suggest that adaptation to cave environments may have occurred either once or multiple times during the evolutionary history of this species. If the latter is true, the unique phenotypes of different cave-dwelling populations may result from convergence of form, and different genetic changes and developmental processes may have similar morphological consequences. Here we report an analysis of variation in the mitochondrial NADH dehydrogenase 2 (ND2) gene among different surface fish and cavefish populations. The results identify a minimum of two genetically distinctive cavefish lineages with similar eyeless phenotypes. The distinction between these divergent forms is supported by differences in the number of rib-bearing thoracic vertebrae in their axial skeletons. The geographic distribution of ND2 haplotypes is consistent with roles for multiple founder events and introgressive hybridization in the evolution of cave-related phenotypes. The existence of multiple genetic lineages makes A. mexicanus an excellent model to study convergence and the genes and developmental pathways involved in the evolution of the eye and pigment degeneration.